Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies. However, since ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu.
Since the development of hybridoma technology in 1975 (Kohler et al., “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256:495-7 (1975)) and, more recently, the development of various in vitro antibody display technologies, (Amstutz et al., “In vitro Display Technologies: Novel Developments and Applications,” Curr Opin Biotechnol 12:400-5 (2001); Dower et al., “In vitro Selection as a Powerful Tool for the Applied Evolution of Proteins and Peptides,” Curr Opin Chem Biol 6:390-8 (2002); Lipovsek et al., “In vitro Protein Evolution by Ribosome Display and mRNA Display,” J Immunol Methods 290:51-67 (2004); Rothe et al., “In vitro Display Technologies Reveal Novel Biopharmaceutics,” FASEB J 20, 1599-610 (2006); and Wark et al., “Latest Technologies for the Enhancement of Antibody Affinity,” Adv Drug Deliv Rev 58:657-70 (2006)) 18 FDA-approved therapeutic antibody products are currently on the market. With many more antibodies in various stages of clinical development, their importance is expected to escalate in the coming years (Holliger et al., “Engineered Antibody Fragments and the Rise of Single Domains,” Nat Biotechnol 23:1126-36 (2005); Hoogenboom, H. R., “Selecting and Screening Recombinant Antibody Libraries,” Nat Biotechnol 23:1105-16 (2005); Reichert et al., “Monoclonal Antibody Successes in the Clinic,” Nat Biotechnol 23:1073-8 (2005); and Hudson et al., “Engineered Antibodies,” Nat Med 9:129-34 (2003)). Recently, innovative recombinant DNA techniques, such as chimerization and humanization, have opened the door to molecular reformatting of naturally produced full-length antibodies into smaller synthetic fragments. These formats exhibit many superior biophysical and biochemical properties and can typically be produced more efficiently and economically (Holliger et al., “Engineered Antibody Fragments and the Rise of Single Domains,” Nat Biotechnol 23:1126-36 (2005)). One such format, the single-chain variable fragment (scFv), consists of covalently linked variable domains (VH and VL) that retain antigen-binding specificity and offers a more suitable format for expression and protein engineering in bacteria and yeast.
The scFv also shows great promise for binding and inactivating target antigens in an intracellular compartment such as the cytoplasm (Biocca et al., “Expression and Targeting of Intracellular Antibodies in Mammalian Cells,” EMBO J 9:101-8 (1990) and Biocca et al., “Intracellular Immunization with Cytosolic Recombinant Antibodies,” Biotechnology (N Y) 1:396-9 (1994)). In principle, the binding properties exhibited by monoclonal antibodies in the extracellular environment should be transferable to the inside of a living cell using intracellularly expressed scFvs, commonly referred to as intrabodies. However, despite the promise of intrabodies, cytoplasmic expression of scFvs is generally confronted with difficulties concerning stability, solubility, and aggregation. The primary reason for these difficulties is that the two conserved intradomain disulfide bonds found in scFvs cannot form under the reducing conditions of the cytoplasm. As disulfide bridges are known to contribute ˜5 kcal/mol to the overall stability of an scFv (Frisch et al., “Contribution of the Intramolecular Disulfide Bridge to the Folding Stability of REIv, the Variable Domain of a Human Immunoglobulin Kappa Light Chain,” Fold Des 1:431-40 (1996)), lack of disulfide bonds typically results in scFv destabilization (Proba et al., “A Natural Antibody Missing a Cysteine in VH: Consequences for Thermodynamic Stability and Folding,” J Mol Biol 265:161-72 (1997)), decreased intracellular solubility (Martineau et al., “Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm,” J Mol Biol 280:117-27 (1998)), limited half-life (Cattaneo et al., “The Selection of Intracellular Antibodies,” Trends Biotechnol 17:115-21 (1999)), and loss of activity (Martineau et al., “Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm,” J Mol Biol 280:117-27 (1998)). Furthermore, the most commonly used approaches for selecting antibodies in the laboratory, including, for example, cell surface display and phage display, yield scFvs that are typically non-functional in the reducing cytoplasm (Visintin et al, “Selection of Antibodies for Intracellular Function Using a Two-Hybrid in vivo System,” Proc Natl Aced Sci USA 96:11723-8 (1999)) likely due to the fact that the expression and isolation process occurs under non-reducing conditions.
Plückthun and coworkers elegantly demonstrated that in vitro ribosome display (Hanes et al., “In vitro Selection and Evolution of Functional Proteins by Using Ribosome Display,” Proc Natl Acad Sci USA 94:4937-42 (1997) and Mattheakis et al., “An in vitro Polysome Display System for Identifying Ligands from Very Large Peptide Libraries,” Proc Natl Acad Sci USA 91:9022-6 (1994)), whereby stabilized antibody, ribosome, and mRNA (ARM) complexes are generated entirely in vitro, can be used for isolating scFvs that are stable under reducing conditions. This was achieved simply by altering the redox potential of the buffer in which the folding of the displayed protein occurred (Jermutus et al., “Tailoring in vitro Evolution for Protein Affinity or Stability,” Proc Natl Acad Sci USA 98:75-80 (2001)). However, this strategy required five rounds of mutagenesis and selection. Furthermore, numerous successes notwithstanding (Lipovsek et al., “In vitro Protein Evolution by Ribosome Display and mRNA Display,” J Immunol Methods 290:51-67 (2004)), in vitro ribosome display can be limited in usefulness, because: (i) efficient in vitro translation and stalling can be technically challenging; (ii) concentrations of cellular factors that may be required for efficient scFv folding differ from concentrations found in vivo; and (iii) in vivo verification is ultimately needed to ensure that any functional improvements discovered in vitro are reproducible inside host cells, where the scFv will be expressed for either in vivo applications or for manufacturing.
The present invention is directed to overcoming these and other deficiencies in the art.